PROJECT SUMMARY/ABSTRACT The long term goal of this project is to elucidate the composition, architecture, and biophysical properties of heterochromatin, and to understand how they contribute to nuclear functions. Heterochromatin is enriched in repeated DNAs, is concentrated in pericentromeric and telomeric regions, and forms a distinct and dynamic 3D domain inside nuclei. Heterochromatin is required for normal sister chromosome pairing and segregation, nuclear architecture, recombination suppression, transposon silencing, and gene silencing. Heterochromatin recruitment is regulated by epigenetic components and mechanisms, specifically di- and tri- methylation of histone H3 lysine 9 (H3K9me2/3) by specific methyltransferases. Heterochromatin Protein 1 (HP1) binds this `mark' and recruits many proteins and complexes to the heterochromatin. We currently lack a clear understanding of the fine structure and organization of the heterochromatin domain, and the biophysical properties responsible for its functions and behaviors. Our preliminary studies in Drosophila have revealed unexpected structural complexity and biophysical properties of heterochromatin that raise questions about our current understanding of the structure and function of this domain, and suggest that heterochromatin may form and function through biophysical mechanisms that have not been associated with chromatin structure and function. In particular, our findings led to the novel hypothesis that the heterochromatin domain forms through a phase separation mechanism, which has recently been shown to compartmentalize functional molecular networks into structures that lack constraining membranes, but has not until now been applied to chromatin domains. We will capitalize on these novel findings and apply advanced imaging, epigenomics, biochemical and biophysical approaches to elucidate: 1) the structural, biochemical and biophysical properties of the heterochromatin domain, 2) the components and mechanisms responsible for heterochromatin formation, and 3) the ways that heterochromatin substructure and biophysical properties contribute to nuclear and organismal functions. Testing the phase separation hypothesis will elucidate important information about the organization and function of heterochromatin in cells and animals, offering the potential of providing a paradigm-shifting foundation for understanding how other chromatin domains form and function. In addition, defective heterochromatin produces genome instability and altered gene expression, contributing to cancer, birth defects, and aging. Understanding how human diseases and conditions alter the biophysical properties that underlie heterochromatin formation and function will ultimately impact the approaches to their diagnosis and treatment.